Abstract Polyploidy (whole genome duplication) is a fundamental aspect of cardiac biology. Metazoan heart structures, from insects to humans, undergo ploidy increases during development and in response to stress and injury. Polyploidy has many functions, from increases in cell size (hypertrophy) and transcriptional capacity to protection from infection and oxidative stress. However, the specific role of developmental or physiological polyploidy in cardiac organogenesis, function, and injury response is unknown. Polyploidy is considered a possible barrier to cardiac proliferation and therapeutic regeneration as ploidy increases coincide with the switch from proliferative to ploidy-increasing hypertrophic tissue repair programs in mammalian development. Thus, by improving our understanding of the significance and regulation of cardiac polyploidy, we can better devise strategies to improve the function of injured hearts. This proposal will develop the Drosophila heart as a model to uncover the conserved yet enigmatic role of polyploidy in heart development and tissue repair. As in mammals, cardiomyocytes of the simple, tubular Drosophila heart are known to polyploidize during development and the adult fly heart can undergo hypertrophy. However, little is known about when Drosophila cardiac polyploidization occurs, its regulation and function, and the interplay between physiological and injury-induced polyploidy. The polyploidization of fly cardiomyocytes will be characterized and perturbed during development and after injury. In Aim 1, the timing and extent of polyploidization during development will be determined. Then, polyploidization will be perturbed and heart function assayed to uncover physiological roles in the heart. Lastly, the gene expression profiles of polyploid and mutant diploid cardiomyocytes will be compared to determine polyploidization-induced genes. In Aim 2, the effect of ploidy on tissue injury responses will be studied. Injury before and after cardiac polyploidization will determine if a developmental switch occurs between proliferative and hypertrophic tissue repair programs. To accomplish this Aim, cardiomyocytes will be genetically ablated and resulting cardiomyocyte proliferation and hypertrophy measured using cell counts and cell cycle markers. Next, ablation experiments will be conducted in mutant diploid and precociously polyploid cardiomyocytes to examine the effect of polyploidy on tissue injury repair. Finally, gene expression changes at a temporal switch in tissue injury repair responses will be compared to find regulators of distinct cardiac injury responses. The above proposed experiments will establish a genetically tractable model with conserved features of cardiac development to uncover the long-appreciated but poorly-understood role of polyploidy in cardiac biology.